The Effect And Mechanisms Of Necrostatin-1 On Hippocampal Neurons After Status Epilepticus In Mice

Objective This investigation was performed to examine the effect of specific necroptosis inhibitor Necrostatin-1 on hippocampal neurons after status epilepticus(SE)induced by kainic acid in mice,and explore the role of Necrostatin-1 on the other two types of programmed cell death(apoptosis,autophagy) in a mouse SE mode.Methods 144 ICR male mice(weighing 25–30g) were randomly divided into three groups: sham control, status epilepticus,and Nec-1 group.To establish animal model of status epilepticus,12mg/kg kainic acid was administered to mice by intraperitoneal injection.The dissolvant of Necrostatin-1 was pretreated with a single intracerebroventricular injection in right hemisphere 15 min before SE. Hematoxylin and Eosin(HE) staining was used to observe the pathological changes in hippocampus CA1?CA3 area. In addition, immunohistochemistry and western blot were used to detect the expression of RIPK1 protein expression at 12h?24h?48h after SE in mice, respectively. In order to investigate the effects of Nec-1 on autophagy and apoptosis in our SE model, we tested the apoptotic-associated proteins( cleaved caspase-3, Bcl-2)and autophagic-associated protei(LC3-II, Beclin-1) expression in hippocampus tissue 24 h after SE in mice.Results(1)To observe by HE staining:about 24 h after the onset of SE, the cells distribution was scattered and cavitation existed in some endochylema, Some neuron swelled and stained uneveuly, especially in CA3 region;Sham group: in the hippocampal CA1, CA3 region, the arrangement and structure of hippocampal neurons remains normal; Nec-1 group:Compared with SE groups, necrostatin-1 pre-treatment can significantly reduced necrotic death of neurons.(2)IHC and western blot analysis showed that,compared with the Sham group, the expression of RIPK1 protein in the SE group was markedly increased since 12 h after SE,and reached the peak at 24h(P<0.05),decreased at 48 h. In contrast, Nec-1 pre-treatment can gradully down-regulated RIPK1 protein level after SE(P<0.05).(3)Western blot showed that Nec-1 pre-treatment suppressed autophagic-associated proteins(LC3-II, Beclin-1) and apoptotic-associated protein(cleaved caspase-3) at 24 h time point after SE(p<0.05). In addition, Nec-1 treatment enhanced anti-apoptotic protein level of Bcl-2 and decreased the Beclin-1/Bcl-2 ratio at 24 h time point after SE(p<0.05).Conclusion Our results suggest that the necroptosis specific inhibitor necrostatin-1 has a significant protection on hippocampal neuronal injury after status epilepticus induced by kainic acid.The neuroprotective mechanisms of Nec-1 may not only be associated with the inhibition of necroptosis,but also with the suppression of apoptosis and autophagy.